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Frontiers in Bioengineering and... 2020P3 has been well-engineered as a platform organism for biologicals production due to enhanced shikimate pathway and excellent physiological and genetic characteristics....
P3 has been well-engineered as a platform organism for biologicals production due to enhanced shikimate pathway and excellent physiological and genetic characteristics. Gentisate displays high antiradical and antioxidant activities and is an important intermediate that can be used as a precursor for drugs. Herein, a plasmid-free biosynthetic pathway of gentisate was constructed by connecting the endogenous degradation pathway from 3-hydroxybenzoate in for the first time. As a result, the production of gentisate reached 365 mg/L from 3-HBA via blocking gentisate conversion and enhancing the gentisate precursors supply through the overexpression of the rate-limiting step. With a close-up at the future perspectives, a series of bioactive compounds could be achieved by constructing synthetic pathways in conventional to establish a cell factory.
PubMed: 33553126
DOI: 10.3389/fbioe.2020.622226 -
Metabolic Engineering Jul 2023The emergence of next-generation sequencing (NGS) technologies has made it possible to not only sequence entire genomes, but also identify metabolic engineering targets...
The emergence of next-generation sequencing (NGS) technologies has made it possible to not only sequence entire genomes, but also identify metabolic engineering targets across the pangenome of a microbial population. This study leverages NGS data as well as existing molecular biology and bioinformatics tools to identify and validate genomic signatures for improving phenazine biosynthesis in Pseudomonas chlororaphis. We sequenced a diverse collection of 34 Pseudomonas isolates using short- and long-read sequencing techniques and assembled whole genomes using the NGS reads. In addition, we assayed three industrially relevant phenotypes (phenazine production, biofilm formation, and growth temperature) for these isolates in two different media conditions. We then provided the whole genomes and phenazine production data to a unitig-based microbial genome-wide association study (mGWAS) tool to identify novel genomic signatures responsible for phenazine production in P. chlororaphis. Post-processing of the mGWAS analysis results yielded 330 significant hits influencing the biosynthesis of one or more phenazine compounds. Based on a quantitative metric (called the phenotype score), we elucidated the most influential hits for phenazine production and experimentally validated them in vivo in the most optimal phenazine producing strain. Two genes significantly increased phenazine-1-carboxamide (PCN) production: a histidine transporter (ProY_1), and a putative carboxypeptidase (PS__04251). A putative MarR-family transcriptional regulator decreased PCN titer when overexpressed in a high PCN producing isolate. Overall, this work seeks to demonstrate the utility of a population genomics approach as an effective strategy in enabling the identification of targets for metabolic engineering of bioproduction hosts.
Topics: Pseudomonas chlororaphis; Metagenomics; Genome-Wide Association Study; Pseudomonas; Phenazines; Bacterial Proteins
PubMed: 37369325
DOI: 10.1016/j.ymben.2023.06.008 -
Micromachines Jul 2019Bacterial viruses or phages have great potential in the medical and agricultural fields as alternatives to antibiotics to control nuisance populations of pathogenic...
Bacterial viruses or phages have great potential in the medical and agricultural fields as alternatives to antibiotics to control nuisance populations of pathogenic bacteria. However, current analysis and purification protocols for phages tend to be resource intensive and have numbers of limitations, such as impacting phage viability. The present study explores the potential of employing the electrokinetic technique of insulator-based dielectrophoresis (iDEP) for virus assessment, separation and enrichment. In particular, the application of the parameter "trapping value" () is explored as a standardized iDEP signature for each phage species. The present study includes mathematical modeling with COMSOL Multiphysics and extensive experimentation. Three related, but genetically and structurally distinct, phages were studied: phage SPN3US, phage ϕKZ and phage 201ϕ2-1. This is the first iDEP study on bacteriophages with large and complex virions and the results illustrate their virions can be successfully enriched with iDEP systems and still retain infectivity. In addition, our results indicate that characterization of the negative dielectrophoretic response of a phage in terms of could be used for predicting individual virus behavior in iDEP systems. The findings reported here can contribute to the establishment of protocols to analyze, purify and/or enrich samples of known and unknown phages.
PubMed: 31277396
DOI: 10.3390/mi10070450 -
Scientific Reports Nov 2023Biocontrol agents are safe and effective methods for controlling plant disease pathogens, such as Fusarium solani, which causes dry wilt, and Pectobacterium spp.,...
Biocontrol agents are safe and effective methods for controlling plant disease pathogens, such as Fusarium solani, which causes dry wilt, and Pectobacterium spp., responsible for potato soft rot disease. Discovering agents that can effectively control both fungal and bacterial pathogens in potatoes has always presented a challenge. Biological controls were investigated using 500 bacterial strains isolated from rhizospheric microbial communities, along with two promising biocontrol strains: Pseudomonas (T17-4 and VUPf5). Bacillus velezensis (Q12 and US1) and Pseudomonas chlororaphis VUPf5 exhibited the highest inhibition of fungal growth and pathogenicity in both laboratory (48%, 48%, 38%) and greenhouse (100%, 85%, 90%) settings. Q12 demonstrated better control against bacterial pathogens in vivo (approximately 50%). Whole-genome sequencing of Q12 and US1 revealed a genome size of approximately 4.1 Mb. Q12 had 4413 gene IDs and 4300 coding sequences, while US1 had 4369 gene IDs and 4255 coding sequences. Q12 exhibited a higher number of genes classified under functional subcategories related to stress response, cell wall, capsule, levansucrase synthesis, and polysaccharide metabolism. Both Q12 and US1 contained eleven secondary metabolite gene clusters as identified by the antiSMASH and RAST servers. Notably, Q12 possessed the antibacterial locillomycin and iturin A gene clusters, which were absent in US1. This genetic information suggests that Q12 may have a more pronounced control over bacterial pathogens compared to US1. Metabolic profiling of the superior strains, as determined by LC/MS/MS, validated our genetic findings. The investigated strains produced compounds such as iturin A, bacillomycin D, surfactin, fengycin, phenazine derivatives, etc. These compounds reduced spore production and caused deformation of the hyphae in F. solani. In contrast, B. velezensis UR1, which lacked the production of surfactin, fengycin, and iturin, did not affect these structures and failed to inhibit the growth of any pathogens. Our findings suggest that locillomycin and iturin A may contribute to the enhanced control of bacterial pectolytic rot by Q12.
Topics: Solanum tuberosum; Tandem Mass Spectrometry; Bacillus; Anti-Bacterial Agents; Bacteria; Plant Diseases
PubMed: 37925555
DOI: 10.1038/s41598-023-46672-1 -
Frontiers in Microbiology 2023Morel mushroom ( spp.) is a rare edible and medicinal fungus distributed worldwide. It is highly desired by the majority of consumers. Bacterial diseases have been...
Morel mushroom ( spp.) is a rare edible and medicinal fungus distributed worldwide. It is highly desired by the majority of consumers. Bacterial diseases have been commonly observed during artificial cultivation of . Bacterial pathogens spread rapidly and cause a wide range of infections, severely affecting the yield and quality of . In this study, two strains of bacterial pathogens, named M-B and M-5, were isolated, cultured, and purified from the tissues of the infected . Koch's postulates were used to determine the pathogenicity of bacteria affecting , and the pathogens were identified through morphological observation, physiological and biochemical analyses, and 16S rRNA gene sequence analysis. Subsequently, the effect of temperature on the growth of pathogenic bacteria, the inhibitory effect of the bacteria on on plates, and the changes in mycelial morphology of mycelium were analyzed when mycelium was double-cultured with pathogenic bacteria on plates. The results revealed that M-B was subsp. and M-5 was . Strain M-B started to multiply at 10-15°C, and strain M-5 started at 15-20°C. On the plates, the pathogenic bacteria also produced significant inhibition of mycelium, and the observation of mycelial morphology under the scanning electron microscopy revealed that the inhibited mycelium underwent obvious drying and crumpling, and the healthy mycelium were more plump. Thus, this study clarified the pathogens, optimal growth environment, and characteristics of bacterial diseases, thereby providing valuable basic data for the disease prevention and control of production.
PubMed: 38029130
DOI: 10.3389/fmicb.2023.1231353 -
Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis.Microbial Biotechnology Sep 2022Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently...
Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently available genome-editing methods for P. chlororaphis are based on homologous recombination (HR)-dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ-Red-like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time-consuming, laborious and inefficient. Here, we established a CRISPR-assisted base-editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced-specificity Cas9 nickase (eSpCas9pp ) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO-inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR-based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non-traditional bacterial hosts.
Topics: Animals; CRISPR-Cas Systems; DNA; Deoxyribonuclease I; Gene Editing; Genome, Bacterial; Pseudomonas chlororaphis; Rats
PubMed: 35575623
DOI: 10.1111/1751-7915.14075 -
Frontiers in Microbiology 2022Currently, plant growth-promoting rhizobacteria (PGPR) microbial inoculants are heavily used in agricultural production among which sp. and sp. are two excellent...
Currently, plant growth-promoting rhizobacteria (PGPR) microbial inoculants are heavily used in agricultural production among which sp. and sp. are two excellent inoculum strains, which are widely used in plant growth promotion and disease control. However, few studies have been conducted on the combined use of the two bacteria. The aim of this study was to investigate the effects of co-inoculation of these two bacteria on soybean [ (L.) ] growth and physiological indexes and further study the effect of microbial inoculants on native soil bacterial communities and plant endophyte microbiota, especially microorganisms in rhizosphere and root. A pot experiment was conducted and four treatments were designed: group without any strain inoculant (CK); group inoculated with H1 inoculant (J); group inoculated with Y1 inoculant (Y) and group inoculated with equal volume of H1 inoculant and Y1 inoculant (H). Compared with CK, the three inoculant groups J, Y, and H exhibited improved soybean growth and physiological indexes, and group H was the most significant ( < 0.05). In terms of rhizosphere bacterial community structure, the relative abundance of native (9.31%) was higher in the H group than in the J (6.07%), Y (3.40%), and CK (5.69%) groups, which has potential value of disease suppression. Besides, compared with bacterial communities of the other three groups in soybean roots, group H increased the abundance of beneficial bacterial community for the contents of , , and significantly increased ( < 0.05). In conclusion, we found that the composite inoculum of H1 and Y1 could effectively promote soybean growth, increase yield and improve the beneficial bacterial community in root and rhizosphere and have certain value for soil improvement.
PubMed: 36699592
DOI: 10.3389/fmicb.2022.1079348 -
Antibiotics (Basel, Switzerland) Jun 2021Blood-borne therapeutic phages and phage capsids increasingly reach therapeutic targets as they acquire more persistence, i.e., become more resistant to non-targeted...
Blood-borne therapeutic phages and phage capsids increasingly reach therapeutic targets as they acquire more persistence, i.e., become more resistant to non-targeted removal from blood. Pathogenic bacteria are targets during classical phage therapy. Metastatic tumors are potential future targets, during use of drug delivery vehicles (DDVs) that are phage derived. Phage therapy has, to date, only sometimes been successful. One cause of failure is low phage persistence. A three-step strategy for increasing persistence is to increase (1) the speed of lytic phage isolation, (2) the diversity of phages isolated, and (3) the effectiveness and speed of screening phages for high persistence. The importance of high persistence-screening is illustrated by our finding here of persistence dramatically higher for coliphage T3 than for its relative, coliphage T7, in murine blood. Coliphage T4 is more persistent, long-term than T3. phage 201phi2-1 has relatively low persistence. These data are obtained with phages co-inoculated and separately assayed. In addition, highly persistent phage T3 undergoes dispersal to several murine organs and displays tumor tropism in epithelial tissue (xenografted human oral squamous cell carcinoma). Dispersal is an asset for phage therapy, but a liability for phage-based DDVs. We propose increased focus on phage persistence-and dispersal-screening.
PubMed: 34208477
DOI: 10.3390/antibiotics10060723 -
Microorganisms Oct 2022Grapevine trunk diseases (GTDs) are caused by multiple unrelated fungal pathogens, and their management remains difficult worldwide. Biocontrol is an attractive and...
Grapevine trunk diseases (GTDs) are caused by multiple unrelated fungal pathogens, and their management remains difficult worldwide. Biocontrol is an attractive and sustainable strategy given the current need for a cleaner viticulture. In this study, twenty commercial vineyards were sampled across California to isolate endophytic and rhizospheric bacteria from different grapevine cultivars with the presence and absence of GTD symptoms. A collection of 1344 bacterial isolates were challenged in vitro against and , from which a subset of 172 isolates exerted inhibition levels of mycelial growth over 40%. Bacterial isolates were identified as ( 154), spp. ( 12), ( 2) and others that were later excluded ( 4). Representative isolates of , and were challenged against six other fungal pathogens responsible for GTDs. Mycelial inhibition levels were consistent across bacterial species, being slightly higher against slow-growing fungi than against Botryosphaeriaceae. Moreover, agar-diffusible metabolites of strongly inhibited the growth of and , at 1, 15, and 30% /. The agar-diffusible metabolites of and , however, caused lower inhibition levels against both pathogens, but their volatile organic compounds showed antifungal activity against both pathogens. These results suggest that , and constitute potential biocontrol agents (BCAs) against GTDs and their application in field conditions should be further evaluated.
PubMed: 36296311
DOI: 10.3390/microorganisms10102035 -
Cell Reports Jul 2021Bacterial communities are in a continuous adaptive and evolutionary race for survival. In this work we expand our knowledge on the chemical interplay and specific...
Bacterial communities are in a continuous adaptive and evolutionary race for survival. In this work we expand our knowledge on the chemical interplay and specific mutations that modulate the transition from antagonism to co-existence between two plant-beneficial bacteria, Pseudomonas chlororaphis PCL1606 and Bacillus amyloliquefaciens FZB42. We reveal that the bacteriostatic activity of bacillaene produced by Bacillus relies on an interaction with the protein elongation factor FusA of P. chlororaphis and how mutations in this protein lead to tolerance to bacillaene and other protein translation inhibitors. Additionally, we describe how the unspecific tolerance of B. amyloliquefaciens to antimicrobials associated with mutations in the glycerol kinase GlpK is provoked by a decrease of Bacillus cell membrane permeability, among other pleiotropic responses. We conclude that nutrient specialization and mutations in basic biological functions are bacterial adaptive dynamics that lead to the coexistence of two primary competitive bacterial species rather than their mutual eradication.
Topics: Adaptation, Physiological; Alleles; Anti-Infective Agents; Bacillus; Bacterial Proteins; Drug Resistance, Bacterial; Microbial Sensitivity Tests; Mutation; Permeability; Pseudomonas
PubMed: 34320359
DOI: 10.1016/j.celrep.2021.109449